The utilization of adult stem cells for organ renewal and repair is an important goal in regenerative medicine. Tooth bioengineering is of great interest, because dental decay and tooth loss constitute an important public health issue. Additionally, tooth anomalies are common in many craniofacial syndromes, and the easy accessibility of the oral cavity makes teeth an excellent test case for organ replacement. A thorough understanding of the molecular and cellular processes that drive tooth renewal and regeneration will be crucial to efforts to build and repair teeth. We are using the mouse incisor as a model for understanding the mechanisms that underlie the ability of stem cells to contribute to renewal of dental tissues, because this remarkable tooth grows continuously due to the presence of adult stem cells. Our preliminary data indicate that the Hippo pathway, via its two effectors, Yes-associated Protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), regulates the incisor stem cell niche. Importantly, our initial studies suggest that YAP/TAZ in the incisor are regulated by mechanical cues; this important regulatory interaction has been understudied in adult stem cell biology. Thus, the rodent incisor provides a unique in vivo platform to study how the physical environment regulates YAP/TAZ, which will not only move the field of Hippo signaling forward but also lay the foundation for developing stem cell-based therapies in the treatment of dental diseases. In this application, we propose to test the hypothesis that mechanical signals, mediated by small Rho GTPases, can modulate YAP/TAZ activity, which in turn control key processes in stem cell biology. In the first Aim, we will determine the effect of Yap/Taz and Lats1/2 loss-of-function in the mouse incisor. Aim 2 will address how mechanical forces and small Rho GTPases regulate YAP/TAZ activity. A pilot RNAi screen will also be carried out to identify novel upstream mechano-regulators of YAP/TAZ. Aim 3 will address the role of the mTor pathway downstream of YAP/TAZ in the incisor stem cells. Together, these experiments will elucidate a linear mechanistic pathway that will help us to understand how Hippo signaling and YAP/TAZ mediate mechanical signals to regulate adult incisor stem cells in mice, and they will yield findings that will be of general interest to both dental researchers and to the stem cell and regenerative medicine communities. The results of these studies will advance our understanding of how nature normally uses stem cells in dental regeneration and enhance our ability to modulate both the physical and biochemical environments in order to generate dental stem cells for translational purposes.